Sodium reabsorption in the distal tubule of the kidney is essential for maintenance of extracellular volume and blood pressure. Aldosterone, vasopressin and insulin stimulate sodium reabsorption by upregulating the activity of ENaC. There is growing evidence that sgkl (serum- and glucocorticoid-induced kinase) mediates the effects of these hormones; however, the signaling pathways employed by sgkl and the molecular mechanisms that modify ENaC activity have not been elucidated. Our working hypothesis stems from a model in which sgkl localizes predominantly at the basolateral membrane of epithelial cells in close proximity with incoming agonists such as insulin or AVP. Phosphorylation of sgkl by these and/or other stimuli to date unknown triggers a signal cascade that reaches the apical membrane to increase the abundance and/or activity of ENaC. The specific aims of this grant proposal are to: 1) Identify the mechanism underlying the effects of sgkl on ENaC in particular, to investigate changes in the traffic of channels, insertion and/or retrieval from the apical membrane, and changes in channel kinetics. 2) Determine the contribution of sgkl to aldosterone, vasopressin and insulin responses, and 3) To identify molecules that by associating with sgkl tether it to membranes, modulate its activity or constitute substrates of the sgkl-signaling cascade. We have developed tools and designed biochemical (turnover of ENaC subunits, phosphorylation assays, modified Ras recruitment system) and electrophysiological (patch-clamp studies and blocker-induced noise analysis) experiments to be conducted in vitro (A6 cell lines modified to express various forms of sgkl and siRNAs under the control of tetracycline) and in rat tissues that together will elucidate the proposed aims. The central role of sgkl in the integration of many signaling pathways makes it a candidate to mediate sodium retention associated with hypertensive disorders. Therefore, understanding the molecular mechanisms of this signaling pathway has potential implications for the diagnosis and development of new treatment strategies for the correction of high blood pressure.